The Role of Mast Cells in Tumor Microenvironment, Tumorigenesis, and Cancer Pain
Throughout the process of tumorigenesis, disease progression, and metastasis, the microenvironment of the local host tissue is an active participant and determines the extent of cancer cell proliferation, angiogenesis, invasion, and survival. The role of mast cells in the tumorigenesis of cancers is not well understood, however it is hypothetically possible that mast cell activation facilitates the growth and spread of some cancers by producing molecules that enhance tumor invasiveness. For example, mast cells have been directly linked to the development of pancreatic cancer tumorigenesis in mouse models, showing that high levels of mast cell infiltration into the tumor microenvironment was predictive of poor clinical outcome, although the exact mechanism by which mast cells contribute to pancreatic cancer development was not clear [Chang D Z et al., Clin Cancer Res 2011; 17:7015-7023]. Hence, inhibition of mast cell function may prove to be of therapeutic benefit in restraining the growth of cancers for which there is sufficient mast cell involvement.
Exactly which cancers would benefit from targeting mast cell activity is however largely unknown or controversial. There exists conflicting data about whether mast cells benefit or hinder tumorigenesis, depending on the local stromal conditions and if the mediators released facilitate the proliferation of tumor cells or induce the apoptosis of malignant cells [(Theoharides T C, et al., Trends Immunol 2004; 25:235-41); (Samoszuk M, et al., BMC Cancer 2005; 21:121); (Almholt K, et al., Recent Results Cancer Res 2003; 162:31-42); (Gooch J L, et al., Cancer Res 1998; 15:4199-205)]. Moreover, consistent with the dual roles of mast cells in inhibiting or promoting tumor growth, high mast cell numbers have been shown to represent a good prognostic indicator in breast cancer, non-small cell lung carcinoma, and ovarian cancer [(Galinsky D S, et al. Crit Rev Oncol Hematol 2008; 68:115-30); (Ribatti D, et al., Int Rev Cell Mol Biol 2009; 275:89-131)], but they are associated with poor prognosis in skin cancer (both melanoma and nonmelanoma and Merkel cell tumors) [Grimbaldeston M A, et al., Br J Dermatol 2004; 150:895-903); (Grimbaldeston M A, et al., J Invest Dermatol 2000; 115:317-20)], oral squamous cell carcinoma, several types of lymphoma, and prostate cancer [(Galinsky D S, et al., Crit Rev Oncol Hematol 2008; 68:115-30); (Ribatti D, et al., Int Rev Cell Mol Biol 2009; 275:89-131)]. It also remains unclear whether mast cell density or the degree of mast cell activation represents the key consideration in mast cell related symptoms, these two aspects not necessarily correlating with one another [Hermine O, et al., PLoS ONE. 2008; 3:e2266].
Mast cells are associated with diverse disease related pain and are emerging as having a role in cancer pain. Mast cells have been linked to the pathogenesis of pain in conditions for which pain is a predominant symptom but is considered to be out of proportion to the objective pathological findings, i.e. indicating that anatomical abnormalities cannot alone account for the pain; examples include chronic pancreatitis, interstitial cystitis, and irritable bowel syndrome. Each of these conditions has been associated with an increased number of mast cells in the pancreas, bladder, or colon, respectively, as compared with those patients without disease related pain. Although the etiology of cancer pain remains unclear, the current understanding indicates that within the cancer microenvironment, cancer and immune cells produce and secrete mediators that activate and sensitize primary afferent nociceptors. Schmidt at al. reviewed the mechanisms of cancer pain [Schmidt B L, et al., Mol Interv. 2010 June; 10(3):164-78], summarizing the symptoms experienced by the cancer patient as being a consequence of cellular, tissue, and systemic changes that occur during proliferation, invasion, and metastasis, with the responding immune system also having a clear role in cancer pain.
Thus, although there is evidence for diverse, indirect mast cell involvement in tumorigenesis (i.e. as opposed to mast cells themselves being the proliferating cancer cell) and also cancer pain, its heterogeneous and disparate nature precludes any clear approach as to how targeting mast cell activity could have a therapeutic impact for cancer patients; one preferably manifested as an augmentation of survival time. This is equally true for those cancers having an established association with increased mast cell involvement, such as pancreatic cancer.
Cancer Pain and Pharmacotherapy Pain Control
The etiology of cancer pain is complex and remains poorly understood. Cancer pain can be severe and debilitating, drastically reducing quality-of-life in patients who already have an attenuated life expectancy. Considering in particular pancreatic cancer, abdominal and back pain is a significant complication with nearly 75% of unrespectable pancreatic cancer patients suffering from pain at the time of diagnosis, increasing to more than 90% of patients in advanced disease [Hameed M, et al., Cancers 2011, 3, 43-60]. Pain in pancreatic cancer may be visceral, somatic, or neuropathic in origin and is produced by tissue damage, inflammation, ductal obstruction, and infiltration. Visceral nociceptive pain is caused by damage to the upper abdominal viscera, structures that are particularly sensitive to stretch, ischemia and inflammation, which typically produces a poorly localized, diffuse pain. Somatic and neuropathic pain may arise from tumor extension into the surrounding peritoneum, retroperitoneum, bones and in the latter case, nerves such as the lumbosacral plexus.
Opioid analgesics are commonly used to manage cancer pain, their mechanism of action being to act directly on the central nervous system. However, this can also lead to unwanted side effects, such as constipation, drowsiness, dizziness, breathing problems, and physical or mental dependence. The World Health Organization (WHO) has published a standardized approach for analgesic drug regimens administered for the control of chronic cancer pain in the form of an “analgesic ladder” [Available online: www.who.int/cancer/palliative/painladder/en/ (accessed on 13 Mar. 2012)]. This model recommends that if pain occurs, there should be prompt oral administration of drugs in the following order: “nonopioids such as paracetamol for mild pain; then, as necessary, mild opioids such as codeine for mild to moderate pain; then strong opioids such as morphine for moderate to severe pain, until the patient is free of pain. To calm fears and anxiety, adjuvants drugs should be used. To maintain freedom from pain drugs should be administered on regular schedule, that is every 3-6 hours rather than on-demand”. This stepwise approach is based on the severity of pain and less on the pathophysiologic process of pain, although it has been recommended that to increase the efficacy of available therapeutic modalities, the multiple types of pain generating processes in cancer (visceral, somatic, and neuropathic) should also be taken into consideration [Hameed M, et al., Cancers 2011, 3, 43-60].
Assessment of Cancer Pain and Quality-of-Life in Cancer Patients
In order to assess and record cancer pain the clinician must select appropriate assessment instruments and procedures. However, there is currently no universally accepted cancer pain assessment tool or consensus even on what such a tool should assess. As a consequence there is great diversity of dimensions and items used in the existing tools, which can affect the validity of pain assessment in general and also makes comparisons between studies difficult. Pain assessment tools may be unidimensional or multidimensional. Based upon literature reviews and expert working groups' opinions, it is generally agreed that single item unidimensional tools are among the most frequently used pain assessment tools in cancer patients. Moreover, for simple assessment of changes in pain intensity and for assessment of pain intensity in clinical settings, Visual Analogue Scale (VAS) based tools have been proven to be psychometrically satisfactory [Jensen, M P, et al., J Pain 2003; 4(1): 2e21]. Unidimensional pain assessment tools include the numeric rating scale (0 is “no pain” and 10 is “worst pain imaginable”); a verbal descriptor scale (“no pain,” “mild pain,” “moderate pain,” “severe pain”); or a visual analogue scale (a 100 mm line with anchors such as “no pain” on the left and “worst pain imaginable” on the right) on which the patient indicates the place on the line that best represents the intensity of pain). Each scale has its strengths and weaknesses; however, most self-report measures of pain intensity are strongly related to one another and can be used interchangeably in many situations, especially when clear instructions and an opportunity for practice has been given.
Subjective pain can be categorized into at least four specific factors: pain intensity, pain affect, pain relief, and pain quality [Jensen, M P, et al., In: Chapman C R, Foley K M, eds.: Current and Emerging Issues in Cancer Pain: Research and Practice. New York, N.Y.: Raven Press, 1993, pp. 193-218]. Pain intensity reflects how much a person hurts, and is the most important factor of pain for the purpose of describing the present invention. For a patient, the rating of pain intensity is a magnitude estimation task. Patients are usually able to provide pain intensity estimates relatively quickly, and measures of pain intensity tend to be closely related to one another statistically. Pain intensity can therefore be viewed as a fairly homogeneous dimension that is relatively easy for most people to gauge. The three most commonly used methods for assessing pain intensity are the Verbal Rating Scale, the Visual Analogue Scale (VAS), and the Numerical Rating Scale. Less commonly used measures include the Behavior Rating Scale, the Picture Scale, the Box Scale, and the Descriptor Differential nScale [Jensen, M P, et al., In: Chapman C R, Foley K M, eds.: Current and Emerging Issues in Cancer Pain: Research and Practice. New York, N.Y.: Raven Press, 1993, pp. 193-218].
Visual Analogue Scales are probably the most frequently used instrument for assessment of pain intensity in the setting of treatment related outcome research. VASs consist of a line, usually 10 cm long, whose ends are labeled as the extremes of pain (for example, “no pain” to “pain as bad as it could be”). If a VAS has specific points along the line that are labeled with intensity-denoting adjectives or numbers, it is referred to as a Graphic Rating Scale of Pain Intensity. Patients are simply asked to indicate which point along the line best represents their pain intensity. Usually, the pain assessor allows the patient to practice using the measure to be sure that the assessment task is understood. The distance from the no pain end to the mark made by the patient is that patient's pain intensity score. There is much evidence supporting the validity of VASs of pain intensity. VASs are directly correlated with other self-report measures of pain intensity, as well as to observed pain behavior [see Jensen, 1993 and references contained therein]. Because VASs are usually measured in millimeters they have a large number of response categories, i.e. the scale can be considered as having 101 points, making it potentially more sensitive to changes in pain intensity than measures with limited numbers of response categories. Research indicates that VASs of pain intensity are usually (but not always) more sensitive to treatment change than are 4- or 5-point Verbal Rating Scale.
Multidimensional pain assessment tools (also sometimes referred to as pain assessment questionnaires) provide a measure of clinical pain that captures its sensory, affective and other qualitative components that extends beyond the basic measure of pain intensity. Theoretically, multidimensional tools should be more reliable and therefore potentially more sensitive for detecting changes in pain associated with time or with treatment; however, they are more complex and lengthy to complete than unidimensional tools. Furthermore, because multidimensional pain or quality-of-life (QOL) assessment tools were generally designed to evaluate change of health related QOL in a clinical trial setting, their scores are only informative when used in a comparative setting, i.e. comparing treatment arms, and therefore, a single individual score is not considered to be informative. Examples of the main multidimensional pain or quality-of-life (QOL) assessment tools used in cancer pain assessment include: the European Organization for Research and Treatment of Cancer 30-item core quality-of-life questionnaire (EORTC QLQ C-30); the Brief Pain Inventory (BPI); and the McGill Pain Questionnaire.
Considering pancreatic cancer in particular, then the EORTC multidimensional tool is arguably the most applicable, although this has yet to be proven in practice. The EORTC QLQ C-30 is a 30-item self-reporting questionnaire developed to assess the quality-of-life of cancer patients [Aaronson, N K et al., J Natl Cancer Inst 85(5): 365-76, 1993]. Importantly, it is supplemented by disease specific modules, including a module specific to pancreatic cancer (QLQ-PAN26), which includes 26 items related to disease symptoms, treatment side-effects and emotional issues. The QLQ-C30 questionnaire has been validated but the QLQ-PAN26 module is not yet validated as it still needs to undergo psychometric testing in a large international group of patients.
In general, the added complexity and patient burden associated with implementation of multidimensional tools outweigh its advantages when the objective is to measure cancer pain intensity or classify patients according to this parameter. Hence, unidimensional tools remain the most appropriate pain assessment option available for the purpose of describing pain in the present invention.
Gene Expression Profiling and Identification of Treatment Subpopulations
Alterations in the genome that lead to a variety of chromosomal aberrations are a characteristic of all malignant tumors. In addition to gene mutations, tumor growth is also sustained by an altered level of gene expression. Gene expression profiling is the measurement of the expression (i.e. activity) of thousands of genes simultaneously, to create a global picture of cellular function or a genetic ‘fingerprint’ of a particular physiological/pathological sample. In the context of cancer, gene expression profiling has been used to more accurately classify tumors; furthermore, comparison of expression profiles can identify subpopulations in which genes are consistently up-regulated or down-regulated. Hence, the information derived from gene expression profiling has the potential to make an objective diagnosis, to identify genes that correlate with survival, to provide risk assessment of premalignant lesions, and to predict responses to certain treatments. In the latter example, one can answer questions of direct clinical significance such as the probability of a patient to respond to a drug given said patient's genetic fingerprint.
Pancreatic Cancer Overview
The pancreas contains exocrine cells (involved in the production of enzymes important for food digestion) and endocrine cells (that produce hormones such as insulin). Both exocrine and endocrine cells can form tumors, but those formed by the exocrine pancreas are far more common and are associated with a very poor prognosis. The vast majority of exocrine pancreatic tumors are adenocarcinomas. Tumors of the endocrine pancreas (also known as islet cell tumors) are far less common and mostly benign in nature.
Cancer of the exocrine pancreas (referred to hereafter as pancreatic cancer) is a seriously life threatening condition. In most cases, early stages of the disease are asymptomatic and less than 20% of pancreatic cancers are amenable to surgery. Of those patients undergoing tumor resection, only 20% will survive 5 years. Early diagnosis of pancreatic cancer is difficult because symptoms vary and are nonspecific. Symptoms are primarily caused by mass effect rather than disruption of exocrine or endocrine functions and depend on the tumor's size and location, as well as the presence of metastases. Cancers that begin in the head of the pancreas are near the common bile duct. These cancers can compress the duct while they are still fairly small, which may possibly lead to jaundice and allow these tumors to be found in an earlier stage. Cancers that begin in the body or tail of the pancreas do not compress the duct until they have spread through the pancreas. By this time, the cancer may have also spread beyond the pancreas, frequently the liver, which also leads to jaundice. All symptoms commonly associated to pancreatic cancer can have multiple other causes, further complicating diagnosis with the consequence that pancreatic cancer is frequently diagnosed at an advanced stage. Moreover, invasive and metastatic pancreatic cancers respond poorly to existing treatments in chemotherapy and radiotherapy, with high levels of carbohydrate antigen 19-9 (CA 19-9), and an Eastern Cooperative Oncology Group (ECOG) status ≥2 also being associated with a poor prognosis. Mortality rate remains obstinately high over the past few decades, with patients receiving standard treatment having a median survival after diagnosis of respectively, 3-6 months and 9-12 months for patients with metastatic and locally advanced disease. The overall 5-year survival rate is below 5%.
Treatment of Adenocarcinoma Pancreatic Cancer
Treatment of pancreatic cancer depends on the stage of the cancer, as described in Table 1. When the disease is confined to the pancreas and clearly separated from surrounding blood vessels (i.e. it is local and respectable), the treatment of choice is surgery with postoperative chemotherapy and/or radiation. When the disease encases or compresses surrounding blood vessels or has extended into adjacent structures (i.e., locally advanced and unresectable), chemotherapy and/or radiation is proposed. In rare cases, when the patient responds well to treatment, the tumor may subsequently be surgically resected. When the disease has spread to distant organs (i.e., metastatic), chemotherapy is proposed. In most cases, these treatments do not represent a cure.
TABLE 1Staging and treatment of pancreatic cancerPancreaticcancerMedianStageDescriptioncasesTreatment optionssurvivalLocal orDisease is confined to15%Surgery; postoperative11-18resectablethe pancreas and ischemotherapy and/ormonthsclearly separatedradiation may also befrom surroundingofferedblood vesselsLocallyDisease encases or40%Chemotherapy (most10-12advanced orcompressescommonly gemcitabine-monthsunresectablesurrounding bloodbased) and/or radiation.vessels, or hasIn very rare instances,directly extended intocancers that respond well adjacent structuresto initial treatment maysubsequently besurgically resected.MetastaticEvidence of45%Chemotherapy (most5-7extrapancreaticcommonly gemcitabine-monthsspread to distantbased); investigationalorgans (liver, lungs,trialsetc.)
Chemotherapy may be used in patients with advanced unresectable cancer (locally advanced or metastatic) and in patients with localized disease after surgery or even as a neoadjuvant treatment to shrink the tumor before surgery. For decades, 5-fluorouracil (5-FU) was the most widely used chemotherapeutic agent in metastatic pancreatic cancer until a randomized study showed symptom benefit and prolongation of survival of gemcitabine (Gemzar®, Lilly France), over 5-Fluorouracil (5-FU). Gemcitabine, a nucleoside analogue of cytidine, is now established as the standard systemic treatment for patients with locally advanced, unresectable, or metastatic pancreatic adenocarcinoma. However, the efficacy of gemcitabine as a single agent remains modest, with a median survival of approximately 6 months in randomized trials and a 12-month survival of approximately 20%. The antimetabolite gemcitabine (CAS number 95058-81-4; (4-amino-1-[3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1H-pyrimidin-2-one) replaces cytidine during DNA replication resulting in apoptosis in cancer cells. Gemcitabine has the following formula:

To date, numerous clinical trials have explored the combination of gemcitabine with either cytotoxic and/or biological targeted compounds, however results have almost universally been disappointing, showing little or no benefit compared with gemcitabine monotherapy. The causes of pancreatic cancer are not well understood but as differences between pancreatic cancer cells and normal cells are uncovered, newer drugs are trying to exploit these differences by attacking only specific targets. Thus, attention is increasingly being directed towards the role of growth factors. Several growth factors and their receptors are overexpressed during the progression of pancreatic cancer, such as epithelial growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF). Deregulated expression of cytoplasmic tyrosine kinases has also been associated with poor prognosis and chemoresistance. In particular, gemcitabine resistance in pancreatic cancer is often associated with high expression of focal adhesion kinase (FAK), a protein involved in metastasis; and elevated expression and activity of Src Family Kinases (SFK), including SRC and Lyn, have also been reported in numerous human cancer cell lines and tumor tissues. Moreover, evidence indicates that recruitment of inflammatory cells, including infiltration by mast cells, facilitates the growth and spread of cancer via the production of molecules that enhance tumor invasiveness.
The epidermal growth factor receptor (EGFR) is the target of several drugs under development, including erlotinib (Tarceva®), the combination of which with gemcitabine has been approved as first-line treatment for patients with unresectable pancreatic cancer. This combination was found to modestly extend survival in a clinical trial, with a median OS (6.24 months) 2 weeks longer than for gemcitabine monotherapy (5.91 months), and 1-year survival rate of 23% (c.f. 17% for gemcitabine monotherapy treatment arm; p=0.023) [Moore M J, et al., J Clin Oncol. 2007 May 20; 25(15):1960-6].
A phase 2/3 multicenter randomized trial was carried out to determine the efficacy and safety of a four drug combination chemotherapy regimen (FOLFIRINOX) (consisting of leucovorin calcium, fluorouracil, irinotecan hydrochloride and oxaliplatin) compared with gemcitabine as first-line therapy in patients with metastatic pancreatic cancer [Conroy T, et al., N Engl J Med. 2011 May 12; 364(19):1817-25]. Each of the drugs in this combination is approved by the FDA to treat cancer or conditions related to cancer. Patients who received the Folfirinox regimen lived approximately 4 months longer than patients treated with the current standard of care, gemcitabine (11.1 months compared with 6.8 months). The objective response rate was 31.6% in patients treated with Folfirinox versus 9.4% in patients treated with gemcitabine. Globally, Folfirinox was associated with a survival advantage but also with notable increased toxicity. Moreover, there exist a number of possible population biases to this study design, as well as possible confounding effects from the study design not being blinded. For example, the study design selected only those patients with a good performance status (ECOG status score of 0 or 1), and because of an increased risk of irinotecan-induced toxicity those patients with a high bilirubin level (typically manifested as jaundice and a common diagnostic sign in patients with pancreatic cancer in the head of the pancreas) were excluded. The implication of these treatment management restrictions and the greater toxicity of Folfirinox, as compared with gemcitabine, are to effectively preclude the use Folfirinox for a sizeable proportion of the global pancreatic cancer population, including those with the poorest prognosis who cannot tolerate this regimen. As such, Folfirinox is appropriate as a first-line option for patients with metastatic pancreatic cancer who are younger than 76 years and who have a good performance status, no cardiac ischemia, and normal or nearly normal bilirubin levels.
Masitinib In Vitro (Re)Sensitization of Pancreatic Cancer Cells to Gemcitabine
We previously discovered that the combination of masitinib and gemcitabine (Gemzar®, Eli Lilly and Company), a nucleoside analog, inhibits the growth of human pancreatic adenocarcinoma. Our in vitro and in vivo studies have shown that masitinib:                Sensitized various cancer cell lines to gemcitabine [Thamm D H, et al. 2011 The Veterinary Journal, doi:10.1016/j.tvjl.2011.01.001].        Sensitized gemcitabine-refractory pancreatic cancer cell lines [Humbert M, et al. (2010) PLoS ONE 5(3): e9430. doi:10.1371/journal.pone.0009430].        Demonstrated antiproliferative activity of the masitinib plus gemcitabine combination in a Nog-SCID mouse model of human pancreatic cancer [Humbert M, et al. (2010) PLoS ONE 5(3): e9430. doi:10.1371/journal.pone.0009430].        
These results supported a hypothesis that masitinib can enhance the antiproliferative activity of gemcitabine in vivo, possibly through chemosensitization. This theory was further reinforced by results from a human phase 2 study, in which patients with advanced pancreatic cancer who received a combination of masitinib (9 mg/kg/day) plus gemcitabine showed improved median time to progression compared with patients treated with gemcitabine alone for the overall population [Mitry E, et al, 2010. Cancer Chemotherapy and Pharmacology 66, 395].